Markers and their use in brain injury

ABSTRACT

The invention relates to a combination of biomarkers and their use in brain injury or mild traumatic brain injury (mTBI) detection.

The invention relates to biomarkers and novel biomarkers, their use indiagnostics of brain injury or brain related injuries, in particularmild traumatic brain injury (mTBI), and methods as well as devices forthe detection of same in an individual.

BACKGROUND OF THE INVENTION

Brain injuries have a high incidence worldwide. In particular mildtraumatic brain injury (mTBI) has a significant incidence in the worldand is responsible for high health cost. In contrast to severe TBI, mTBIis not obvious to detect and thus usually a computer tomography (CT)scan is performed before significant brain injury can be ruled in orout.

So far, it is still a challenge identifying which patients with a numberof neurological injuries and in particular a mild traumatic brain injury(mTBI), can be safely sent home. Computer tomography (CT) scan is thusthe main tool today to detect a cerebral lesion in these patients.However, many of the scans are negative and cost-intense. Therefore, inany clinical decision rules for mild TBI, defined as presenting with aGlasgow Coma Score (GCS) (Jennet and Teasdale, check, 1978) of 13-15,rapid and reliable identification of patients with intracranial lesionsis critical to avoid post-traumatic complications and minimize secondarybrain damages (Graham, et al., 1998). Several studies aimed to firstscreen all mild TBI-patients with a simple blood test to reduce thenumber of unnecessary CT scans and discharge patients faster have beenreported (Berger et al., 2007; Poli-de-Figueiredo et al., 2006). In thelast years, especially S100B was extensively investigated as potentialpromising marker for mTBI and is highly promoted by companies.Nevertheless, its clinical utility remains controversial. In mild TBIadults, S100B below a cut-off of 0.10 μg/L was described to allow amaximal reduction of only 30% in CT scans. S100B failed to be a relevantprognostic marker for paediatric TBI patients, estimated only as adjunctin determining children with low-risk TBI (Tavarez et al., 2012).

A mild traumatic brain injury (mTBI, also called concussion, minor headtrauma, and minor brain/head injury) is a type of closed head injury,defined as the result of a blunt trauma or acceleration/decelerationforces causing a brief change in mental status (confusion,disorientation or loss of memory) or loss of consciousness for less than30 minutes. Usually, loss of consciousness is very brief and rangesbetween a few seconds to minutes. Mild TBI remains the biggestpercentage of all closed head, brain injury cases admitted to thehospitals. Currently, the primary criterion for evaluating patients withTBI in clinical setting is the Glasgow Coma Scale (GCS), which assessesthe level of consciousness following TBI. A mild traumatic brain injuryis most likely to be diagnosed only when there is a change in the mentalstatus at the time of injury or hospital admission (the person is dazed,confused, or loses consciousness, GCS score 13-15). In US 10% of headinjury patients are classified at admission as having severe (GSC below8), 10% as moderate (GCS 9-12), and remaining 80% as mild TBI (GCS13-15) (Narayan R K, Michel M E, et al, J Neurotrauma., 2002), Similarproportions are indicated by World Health Organization in Europe, thatestimated 70 to 90% of treated head injuries are classified to presentas mild (Cassidy J D et al. 2004. Journal of Rehabilitation Medicine).It remains a public health problem as 10% of patients with mTBI cansuffer long-term disabilities such as headache, fatigue, difficultythinking, memory problems, attention deficits, mood swings, sleepdisorders, frustration and even epileptic events (Jail° and Narayan,2000; Narayan et al, 2002). Due to the complicated etiology it remainschallenging to identify which patients with mTBI can be safely sent homewithout the need for treatment intervention (Jagoda et al., 2008).Currently to counter-act possible post-traumatic complications andsecondary brain damages mTBI patients are further diagnosed with toolssuch as computerized tomography (CT) scans and magnetic resonanceimaging (where available) (Graham et al., 1998). In the group ofpatients with mTBI only 3 to 19% present with an abnormal CT resultrevealing an acute intracranial lesion in patients (Jagoda et al., 2008;Bazarian et al., 2006; Borg et al., 2004). The other 80%+of these scansshow normal head CT, indicating no complications from injury, and assuch are not cost effective and are time-consuming for both patient andmedical staff.

The use of biomarkers has been proposed as a means to reduce the amountof unnecessary CT scans (Berger et al. 2007; Poli-de-Figueiredo et al.,2006) and for use in decentralized sites where access to CT equipment isabsent. However so far no biomarker-assay is available which gives testresults capable of properly classifying the majority of patients andtherefore useful in serial screening.

As described the method of choice today is a CT scan due to theinsufficient reliability and high percentage of false negative resultswith known biomarkers for TBI detection. One such known biomarker isS100B.

In recent years, S100B has been extensively investigated as a potentialpromising blood marker for mTBI (Ruan S et al., 2009; Goyal et al,2013), Nevertheless, its clinical utility remains controversial. In mTBIadults, S100B below a cut-off of 0.1 μg/L was described to allow amaximal reduction of 30% in CT scans (ref from researchers), S100Bfailed to be a relevant prognostic marker for pediatric TBI patients,estimated only as adjunct in determining children with low-risk TBI(Tavarez et al,, 2012; Filippidis et al., 2010).

S100B is a low-molecular-weight (9-13 kDa), non-ubiquitousCa2+-modulated protein implicated in e,g. regulation of enzymeactivities, dynamics of cytoskeleton elements, cell growth anddifferentiation and Ca2+ homeostasis (Donato R., 2003). In the centralnervous system (CNS) it is primarily found and secreted by glial cells(Donato R., 2003). Due to its involvement in calcium homeostasis it hasneuroprotective function e,g, prevents mitochondrial failure and celldeath in the absence of glucose by increasing cellular calciumconcentrations (Bargeror et al, 1995) or promote neurite outgrowth andastrocytic proliferation (Reeves et al, 1994). Significantly increasedS100B levels are associated with severe TBI and may reflect ongoingstructural damage and cell death after injury (Ingebrigtsen et al. 2002,Missler et al, 1999).

In an injury like mTBI one cannot risk a significant percentage of falsenegatives in view of the detrimental consequences if a patientexhibiting mTBI would be allowed to leave the hospital and sufferserious complications, or even death thereafter due to a wrongdiagnosis. Thus the cut-offs defined for such tests need to be biasedtowards very high specificity (close to100%) which can result in a verylow sensitivity in consequence. This limitation has made the knownindividual biomarkers for mTBI not feasible for routine diagnostics in aclinical setup.

In view of the cost pressure in healthcare and the high cost of a CTscan it is highly desirable and a long felt need to find alternative,reliable and cost-effective routes of classifying a potential mTBIpatient.

Thus one object underlying the present application is to provide foralternative or new feasible biomarkers for the detection or/andclassifying of any brain related traumatic state and in particular formTBI, and for assays and devices useful and reliable therefore and inmTBI diagnostics which can be used in a clinical or non-clinicalcontext, or to improve known approaches to neurological or mTBIscreening and analysis.

SUMMARY OF THE INVENTION

In one aspect the invention provides a method, composition, kit, assayfor the classifying or detection of brain injuries or disorders ordiseases like TBI, transient ischemic attack, brain tumors, seizures,epilepsia, cerebral abscess, encephalopathies and multiple sclerosis byuse of a combination of markers like glutathione S transferase Pi(GSTP), fatty-acid-binding protein (FABP), glial fibrillary acidicprotein (GFAP), neuron-specific enolase (NSE), neuromodulin (GAP43),neurofilament protein H (NFH), neurofilament protein M (NFM),neurofilament protein L (NFL), S100B, Tau, spectrin breakdown products,ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1), vascular celladhesion protein 1 (VCAM), serum amyloid A (SAA), Chernokine (C-C motif)ligand 23 (CCL23), peroxiredoxin 1 to 6 and nucleoside diphosphatekinase (NDKA).

In another aspect the present invention provides a combination of aselection of blood brain biomarkers (GSTP1, H-FABP, VCAM, NDKA)optionally combined to the known (and not sufficiently specific) S100Bfor reliable detection of brain injury like a disease or disorderselected from the group consisting of TBI, transient ischemic attack,brain tumors, seizures, epilepsia, cerebral abscess, encephalopathiesand multiple sclerosis in a sample and in particular for mTBI detection.In particular these biomarkers were combined and adjusted in panelsyielding specificity above 50% at a fixed sensitivity of 95% or 100%.This will allow clinicians and also on site medical emergency staff tobetter manage the detected diseases or disorders and in particular mildTBI patients and therefore potentially reduce CT scans but also theconsequences associated with a delayed diagnosis of brain injury.

In another aspect the invention concerns devices, e.g. biamarker panels,to detect early traumatic brain injury (TBI) lesions better orcomplement S100B to rule out CT scans in mild TBI patients.

In other aspects the invention relates to methods and devices for thedetection of a medical condition in a patient like TBI and mTBI.

In another aspect the invention relates to a method and devices makinguse of an algorithm to detect a medical condition of an individual in asample,

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a comparison of S100B (specificity 37%)-and a combinationof S100B+GSTP+HFABP (specificity 58%) for a 100% sensitivity in a cohortof mTB1 patients. The invention thus can significantly improve thereliability of test results whereby it was not expected that saidcombination could provide for such an improvement and positive testresults,

FIG. 2 shows a comparison of S100B (specificity 37%) and a combinationof S100B+NDKA (specificity 49%) for a 100% sensitivity in a cohort ofmTBI patients.

FIG. 3 shows a comparison of S100B (specificity 37%) and a combinationof S100B+GSTP+HFABP (specificity 61%) for a 95 -100% sensitivity in acohort of mTBI patients.

FIG. 4 shows a comparison of SlOOB (specificity 37%) and a combinationof S100B+1-1FABP (specificity 52%) for a 95 -100% sensitivity in acohort of mTBI patients.

FIG. 5 shows a comparison of S100B (specificity 37%) in a full cohortand a combination of S100B+GSTP+Age (specificity 64%) for a 95 -100%sensitivity in a cohort of mTBI patients. The age group in this case wasdefined in years.

FIG. 6 shows a comparison of S100B (specificity 37%) and VCAM(specificity 40%) for a 100%© sensitivity in a cohort of mTBI patients.

FIG. 7 shows a comparison of S100B (specificity 37%) and a combinationof HFABP+VACM (specificity 59%) for a 100% sensitivity in a cohort ofmTBI patients

The scheme of FIG. 8 shows the advantage of using the biomarkers of theinvention to avoid costly CT scans in a clinical setup.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a method for screening for a disease ordisorder selected from the group consisting of TBI, transient ischemicattack, brain tumors, seizures, epilepsia, cerebral abscess,encephalopathies and multiple sclerosis in a specimen (sample)comprising the steps of using a sample under suitable conditions anddetecting at least two biomarkers under suitable conditions wherein thebiomarkers are selected from the group consisting of glutathione Stransferase Pi (GSTP), fatty-acid-binding protein (FABP), glialfibrillary acidic protein (GFAP), neuron-specific enolase (NSE),neuromodulin (GAP43), neurofilament protein H (NFH), neurofilamentprotein M (NFM), neurofilament protein L (NFL), S100B, Tau, spectrinbreakdown products, ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1),vascular cell adhesion protein 1 (VCAM), serum amyloid A (SAA),Chemakine (C-C motif) ligand 23 (CCL23), peroxiredoxin 1 to 6 andnucleoside diphosphate kinase (NDKA).

The invention has the potential to make a world broad impact on theclinical practice in the management of brain injuries and in particularmTBI. The invention is feasible to provide for a diagnostic panel ofmarkers that can be easily used and are reliable and safe.

The results described below on S100B, GSTP, NDKA, VCAM and H-FABP panelsof at least two of these molecules highlight that a point of care test(PACT) or an array can be readily used for diagnostic purposes.Traumatic brain injury (TBI) is the leading cause of death anddisability in adults younger than 40 years and in children worldwide.Accurate determination of the initial brain damage after brain injury iscrucial in establishing a neurologic prognosis and to balance risks andbenefits of treatment options. The invention advantageously provides forsuch a tool and method.

In a preferred embodiment the invention relates to a method wherein thebiomarkers are selected from S100B, GSTP, HFABP, VCAM and NDKA orfragments, variants or mutants thereof. Particularly useful is thecombination of two or three markers.

Unexpectedly and surprisingly the inventors could show that by acombination of at least two markers of the invention a reliable and easyto use method can be provided to reliably analyse a specimen and therebyrule out brain injury complications in an individual characterized bymTBI and thereby to avoid costly CT scans or even transportation to acentre capable of performing such.

It was not predictable that the markers of the invention and theparticular selection of certain markers or/and the combination ofcertain markers could be applied to provide for a reliable and specificmethod to assay for the brain injuries or disorders or diseases ormedical complications as described herein and particularly TBI and mTBI.The invention will have not only a positive impact on cost in suchanalysis and specifically mTBI analysis but represents also an easy touse and fast method in this medical area.

In particularly the invention is advantageous in that it provides for animproved specificity (>50%) for 100% sensitivity to rule out CT scans inbrain injuries or other complications and mild TBI patients using apanel of at least two markers within S100B, GSTP, HFABP, VCAM and NDKA.

A direct comparison of available state of the art markers with theinvention makes it apparent how advantageous the invention is: with apanel of 3 new biomarkers according to a preferred embodiment of theinvention, whereby sensitivity is 100%, 58% of the mTBI could bedirectly discharged compared with 34% for individual 51008 markeranalysis.

Another advantageous embodiment is a combination with the moleculemarkers as described herein with other markers like age or GCS. Inparticular such an application and use of the markers according to theinvention in a particular patient group characterized by GCS score of 13or more or 15 or more, or in a particular age group, e.g. 60 years ormore, 65 years or more, 70 years or more will yield very positive andhighly reliable test results. More so, in this manner the inventionpositively achieves that reliable test results can be achieved by theuse of less molecular markers which does not only have technicaladvantages but is also advantageous from a cost point of view.

The invention provides the unexpected advantage that a brain injury andthe particular medical complications as described herein can beidentified and screened for not only in a fully equipped hospital but byuse of a simple test device anywhere and without the use of qualifiedmedical personnel,

In the following certain terms of the invention will be defined in moredetail

“Brain injury” is any state of a patient or individual which is thecause of sudden impact on the head or the individual. A particular braininjury is TBI or mTBI.

“TBI” in the sense of the invention is any brain injury caused by atraumatic incident as described above with reference to the prior art.

“Identification” or “identify” or “classify” in the sense of theinvention is the analysis of a sample of an individual to assess whetherthe individual has a brain injury and particularly TBI or mTBI; theidentification of e.g. TBI and mTBI can be verified by use of a CT scanor MRI analysis.

“Diagnostic method” or “diagnostic” in the sense of the invention is anyuseful method with a suitable sequence of method steps for thedetection, visualization and/or quantification of the test resultgenerally known in the art.

“Assay” in the sense of the invention is any method generally known inthe art to detect TBI or mTBI like EL ISA or any other standard methodsfor detection of biomarkers.

“Device” in the sense of the invention is a combination of thebiomarkers or panel of biomarkers according to the invention that can beused to perform an assay for TBI or mTBI detection, Examples are carrierplates, test stripes, biochip arrays or the like known in the art.

“Marker” or “biomarker or “molecular marker or “molecular biomarker inthe sense of the invention is any useful biomarker to detect in a sampleof preferably blood, plasma, saliva, tears, CSF or urine a brain injury,preferably traumatic brain injury (TB!) or other disorders as describedbelow; preferably the combination of at least two markers is suitable todetect mild TBI (mTBI); the markers are used in a suitable assay setupwherein preferably the selectivity is set to 100% and the specificity ispreferably more than 40%, even more preferred more than 50%, more than55%, more than 58%, 60% or 70%.

As a marker in the sense of the invention qualifies any marker of glialcells, neuronal cells, or vascular cells. Preferred markers of theinvention are:

Glutathione S Transferase Pi (GSTP)

Fatty-acid-binding protein (FABP)

Glial fibriliary acidic protein (GFAP)

Neuron-specific enolase (NSE)

Neuromodulin (GAP43)

Neurofilament protein H (NFH)

Neurofilament protein M (NFM)

Neurofilament protein L (NFL)

S100B

TAU

Nucleoside diphosphate kinase (NDKA)

Spectrin breakdown products

Ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1)

Vascular cell adhesion protein-1 (VCAM)

Serum amyloid A (SAA)

Chemokine (C-C motif) ligand 23 (CCL23)

Peroxiredoxin 1 to 6

In addition to the “markers” as described above it is also within thescope of the invention that one, two, three or even more markers can becombined with defining the patient or individual by age or GCS score.Age and GCS can thus be denoted as “marker” in the sense of theinvention. Such markers like age and GCS can also be used in the senseof the invention to define a patient subgroup or subgroup ofindividuals. A preferred age group is below 50, 60, 70 or more than 50,60, 65, 70 years of age. A GCS of 13 to 15 can preferably be used todefine a patient subgroup and can be used in combination with any of theother markers defined herein. In such a manner the individuals can bestratified and patient groups can be formed both to adapt and increasethe test performance or to reduce the markers needed to achieve areliable test result and preferably to adjust the features of thedetection method or the components of a test kit.

A marker “panel” in the sense of the invention is a combination of atleast two biomarkers, preferably two or three markers, used incombination in a suitable setup or device.

“Sensitivity” in the sense of the invention refers to the assay resultof true positives in the analysis of TBI or mTBI. Preferably thesensitivity in the analysis according to the invention is set to 95% to100%, or 100% (i.e. no false negative diagnoses).

“Specificity” in the sense of the invention is the so-called truenegative rate in an assay to identify TBI or mTBI. The specificity ispreferably targeted to be at least 50% and preferably higher, e.g. 58%,60%, 65%, 70%.

A “sample” or “specimen” in the sense of the invention is any fluid ortissue useful for performing an assay or detection method to identifyTB!, preferably mTBI. Preferably the sample is a blood, plasma or urinesample taken from an individual. The sample is treated according togenerally known procedures to keep or make them feasible for the markeranalysis according to the invention.

In the following preferred embodiments of the invention will bedescribed.

In a preferred embodiment the invention relates to a method wherein thesample is blood, plasma, saliva, tears, CSF, or urine. A blood sample isa very easy way of sample collection and thus the method according tothe invention will be readily performed with simple means,

The method according to the invention is can be applied to alldisorders, diseases or medical complications as described herein and isparticularly useful for TBI and in particular for mild TBI (mTBI).

In an alternative embodiment the invention concerns a compositioncomprising or consisting of at least two markers useful for TBIdetection in a sample of an individual wherein the markers are selectedfrom the group consisting of glutathione S transferase Pi (GSTP),fatty-acid-binding protein (FABP), glial fibrillary acidic protein(GFAP), neuron-specific enolase (NSE), neuromodulin (GAP43),neurofilament protein H (NFH), neurofilament protein M (NFM),neurofilament protein L (NFL), S1008, Tau, spectrin breakdown products,ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1), vascular celladhesion protein 1 (VCAM), serum amyloid A (SAA), Chemokine (C-C motif)ligand 23 (CCL23), peroxiredoxin 1 to 6 and nucleoside diphosphatekinase (NDKA).

Said composition advantageously is comprising or consisting of two orthree markers wherein the markers are selected from S100B, GSTP, HFABP,VCAM and NDKA or fragments, variants or mutants thereof.

In an alternative embodiment the invention relates to a kit comprisingor consisting of two or three markers of any of the above markers.

Furthermore, the invention relates to an assay device comprising orconsisting of two or three markers of any of the above markers.Preferably the Assay device comprises or consists of a biochip,biomarker panel on a carrier, or test strip.

In addition to the above described embodiments it will be possible tocombine the method and device of the invention with non-markerobservations on the patient as part of a decision matrix, e.g. braininjury score, pupilar dilation, cognitive tests etc. which will lead toa reliable decision making of hospitalization of an individual orliberating him.

The invention encompasses also further subgroups of marker combinationsbeing advantageous in terms of the test results that can be achieved.These subgroups are a combination of two, three, four or five markersselected from the group consisting of GSTP, FABP, GFAP, NSE, GAP43, NFH,NFM, NFL, S100B, VCAM, SAA, CCL23, peroxiredoxin 1 to 6 and NDKA. Inparticular preferred is a combination of two or three markers of GSTP,FABP, GFAP, NSE, GAP43, NFH, NFM, NFL, S100B, VCAM, SAA, CCL23,peroxiredoxin 1 to 6 and NDKA, more preferably a combination of S1006,VCAM and H-FABP, or GSTP, VCAM and H-FABP, or GSTP, FABP and GFAP, orSAA, VCAM and H-FABP, or peroxiredoxin 1, VCAM and H-FABP, or CCL23,VCAM and H-FABP, or S100B, VCAM and CCL23, or S100B, CCL23 and H-FABP,or NSE, GAP43 and NFH, or NFM, NFL and S1008, or FABP, GFAP and NSE, orGAP43, NFH and NFM, or NFL, S1006 and NDKA, or GAP43, NFH and NFM, orNFL, S1006 and NDKA, or GFAP, NSE and GAP43, or NFH. NFM and NFL.

The invention will be described in more detail in the following exampleswhich are meant to be illustrative without any restriction and whichrepresent preferred embodiments of the invention.

As will be apparent from the experimental part describing the invention,the invention has the advantage that it achieves very reliable testresults. Accordingly in preferred embodiments it provides for a method,composition, kit or assay wherein the sensitivity is more than 95%,preferably 100%, and the specificity is more than 50%, preferably morethan 55%, more preferably more than 60%, and more preferably more than70%.

In an alternative embodiment the invention provides for a method of or asystem for analyzing in a specimen a medical condition wherein a medicaldevice as described herein is applied under appropriate conditions,making use of any of the biomarkers described herein for the analysis ofany of the disorders or diseases of the invention and making use of analgorithm wherein the test results are further defined by way of thealgorithm, e,g, quantified.

The steps of such a method or system are in a preferred embodiment asfollows: The method and system are capable to analyze at least two testresults in a sample of an individual, useful for the diagnosis of amedical condition like brain injury, with the system comprising:

(a) at least two databases comprising:

(i) a first test result collected from a first diagnostic test;

(ii) a second test result collected from a second diagnostic testdifferent than the first diagnostic test;

(iii) optionally, subsequent test result(s) collected from subsequentdiagnostic test(s) different from the previous diagnostic test(s)

(iv) optionally , secondary subject observations or measurements;

(v) one or more diagnostic cut-offs associated with the first diagnostictest, with the second diagnostic test, with subsequent diagnostic tests,and with the subject observations or measurements, wherein such cut-offscollectively integrate to assess probability of brain injury status.

(b) one or more processors operatively encoded to automatically:

(i) apply an interpretation algorithm to generate a subject resultcoordinate based on the database of test results.

(ii) optionally apply a second interpretation algorithm to generate aprobability of error in the subject result coordinate.

EXAMPLES

The following examples are meant to illustrate the invention in moredetail without to be construed as limiting in any sense.

The blood GSTP1, H-FABP, NDKA, VCAM and S100B content of patientspresenting or not cerebral lesions on CT scan with a Glasgow ComaScale >13 and within 6 hours after the onset of TBI were quantified andcompared using ELISA analysis. The study population comprised a total of97 individuals.

ELISA was performed using 96-well homemade assays for GST-Pi and NDKA asdescribed elsewhere (Turek, et al. 2012), H-FABP from Hycult (NL) andCCL23, SAA, peroxiredoxin 1 and S1 00B from Abnova (TW). Each plasmasample were assayed in duplicate and distributed randomly on the plates.

Protein levels were initially expressed in relative fluorescence unit(RFU) and concentrations were calculated using a calibration curveobtained on the same plate with the recombinant proteins. Statisticalanalyses were performed using IBM SPSS Statistics software version19.0.0 (IBM Corporation, NY, USA). To assess the ability of proteins todiscriminate between different populations, non-parametric tests wereperformed. A Wilcoxon matched pairs test was performed for age and sexmatched data and a Mann-Whitney for non-matched data. For datacontaining more than two groups, a one-way ANOVA Kruskal-Wallis test wasused. Receiver Operating Characteristic (ROC) curves analysis wasperformed and cut-off (CO) points obtained from the curves. Optimalthreshold values were chosen to provide the highest specificity for 100%sensitivity. Multivariate logistic regression analysis was used tocompare the values of plasma S100B, H-FABP, GST-Pi, VCAM, CCL2 3, SAA,peroxiredoxin 1 and NDKA levels as CT scan rule out markers.

In these experiments the inventors succeeded in providing a panel (i.e.a small set of two or three) of biomarkers or biomolecules that could beuseful in a clinical setting. We could show that each member of thepanel provides a different angle to the diagnosis and taken togetherthey lead to a more accurate prediction. Each member of the panelfulfils several criteria: firstly it must have a predictive poweritself, i.e. it must be able to distinguish the disease types to acertain extent. Secondly it must be easy to measure with highreproducibility. Thirdly, it should have a central role in thebiological processes that were found by the network analysis.

S100B, H-FABP, GST-Pi, VCAM, CCL2 3, SAA, peroxiredoxin 1 and NDKA wereidentified as particularly useful in such a panel analysis according tothe invention. We developed PanelomiX toolbox, which is able to extractoptimal panels from a small number of molecules and provides a simple,easy to interpret set of threshold rules for disease typeclassification. The rule-based classifier just counts the number ofmolecules whose quantity passes specific threshold values. It mimics theway many clinical scores are built and is therefore easy to understandby people working in a clinical environment. Briefly, the optimizedcut-off values were obtained by iterative permutation-responsecalculations using all available parameters. Each cut off value waschanged iteratively by quantities of 2% increment, and specificity wasdetermined after each iteration until a maximum of specificity wasachieved for 100% sensitivity.

Results

The figures as described above show the results of the specificitycomparison of S100B alone vs. different panels of two or threemolecules/biomarkers of the invention. The sensitivity has been set at100%. It demonstrates that our cohort is comparable to all publishedmTBI cohorts analysing S100B. When sensitivity is set at 100%, a 37% ofspecificity is obtained. GSTP and HFAB have a specificity of 25%, NDKAof 12% and VCAM of 40%. This confirms all previous results on thelimited capacity of S100B alone to rule-out negative CT patients and theneed of additional parameters/biamarkers,

Marker(s) Sensitivity % Specificity % S100B 100 37 GSTP 100 25 HFABP 10025 NDKA 100 12 VCAM 100 40 SAA 100 CCL23 100 Peroxiredoxin 100

When molecules are combined into panels of three molecules, thespecificity increases up to 58% (S100B, HFABP and GSTP) and up to 49%when two molecules are combined (S100B and NDKA).

Marker(s) Sensitivity % Specificity % S100B/HFABP/GSTP 100 58S100B/HFABP/NDKA 100 57 GSTP/HFABP/NDKA 100 51 S100B/VCAM/HFABP 100 60S100B/VCAM/SAA 100 59 S100B/VCAM/CCL23 100 63 S100B/VCAM/Perox 1 100 62S100B/NDKA 100 49 GSTP/HFABP 100 43 HFAB/VACM 100 59 S100B/VACM 100 46S100B/CCL23 100 45 HFABP/CCL23 100 48 VCAM/CCL23 100 44

The results described here on S100B, GSTP, NDKA, VCAM, GCL23, SAA,peroxiredoxin 1 and H-FABP panels of at least two of these molecules ora combination of three as shown highlight that they can be easily usedin a PACT or an array for serial diagnosis.

The combination of a least two out of GSTP1, H-FABP, VCAM, NDKA, CCL23,SAA, peroxiredoxin 1 and S100B in panels gives rise to increasedspecificity above 50% for a sensitivity of 100% to rule out CT scans inmild TBI patients.

Accordingly, the surprising and unexpected advantage is an improvedspecificity (>50%) for 100% sensitivity to rule out CT scans in mild TBIpatients using a panel of two or three markers within S100B, GSTP,HFABP, VCAM, CCL23, SAA, peroxiredoxin 1 and NDKA.

The experimental results and the advantages of the invention are alsoapparent from the following tables:

Sensitivity % Specificity % Sensitivity % Specificity % S100B 100 3795-100 37 GSTP 100 25 95-100 36 HFABP 100 25 95-100 25 NDKA 100 1295-100 27 VCAM 100 40 — — CCL23 100 25 95-100 23 SAA 100 23 95-100 20Peroxiredoxin 1 100 20 95-100 22 Marker(s) Sensitivity % Specificity %Sensitivity % Specificity % S100B/HFABP/GSTP 100 58 95-100 61S100B/HFABP/NDKA 100 57 95-100 60 GSTP/HFABP/NDKA 100 51 95-100 52S100B/GSTP/Age — — 95-100 64

Marker(s) Sensitivity % Specificity % Sensitivity % Specificity %S100B/NDKA 100 49 — — S100B/HFABP 95-100 52 GSTP/HFABP 100 43 95-100 49HFABP/VACM 100 59 95-100 62

Results in a patient group which includes only patients with an age ofabove 61 years are depicted in the following:

S100B/GSTP/Age — — 95-100 64

It could also be shown that in said age group/patient population (i.e.patients above 61 years old) the best single biomarker is not anymore6100B but GSTP with a specificity of 50% as depicted below:

Sensitivity % Specificity % Sensitivity % Specificity % GSTP/Age — —95-100 50

1. Method for screening for a disease or disorder selected from thegroup consisting of TBI, transient ischemic attack, brain tumors,seizures, epilepsia, cerebral abscess, encephalopathies and multiplesclerosis in a sample comprising the steps of using a sample undersuitable conditions and detecting at least two biomarkers under suitableconditions wherein the biomarkers are selected from the group consistingof glutathione S transferase Pi (GSTP), fatty- acid-binding protein(FABP), glial fibrillary acidic protein (GFAP), neuron-specific enolase(NSE), neuromodulin (GAP43), neurofilament protein H (NFH),neurofilament protein M (NFM), neurofilament protein L (NFL), S100B,Tau, spectrin breakdown products, ubiquitin carboxyl terminalhydrolase-L1 (UCH-L1), vascular cell adhesion protein 1 (VCAM), serumamyloid A (SAA), Chemokine (C-C motif) ligand 23 (CCL23), peroxiredoxin1 to 6 and nucleoside diphosphate kinase (NDKA).
 2. Method according toclaim 1 wherein one or more biomarkers according to claim 1 is combinedwith a marker which is age or a defined GCS, preferably 13 to
 15. 3.Method according to claim 1 wherein one or more biomarkers according toclaim 1 is used for screening a defined age or age group or a definedGCS group.
 4. Method according to claim 2 wherein the age group has anage of less than 50, less than 60, or more than 60, more than 70, ormore than
 80. Method according to claim 2, 3 or 4 wherein the GCS is atleast 13 or at least
 15. 5. Method according to claim 1 wherein thebiomarker is selected from S100B, GSTP, HFABP, VCAM, serum amyloid A(SAA), Chemokine (C-C motif) ligand 23 (CCL23), peroxiredoxin 1 to 6 andNDKA or fragments, variants or mutants thereof.
 6. Method according toclaim 1 wherein the sample is blood, plasma, urine, saliva, tears(lachrymal fluid), or CSF.
 7. Method according to claim 1 wherein theTBI is mild TBI (mTBI).
 8. Composition comprising or consisting of atleast two markers useful for the detection of a disease or disorderselected from the group consisting of TBI, transient ischemic attack,brain tumors, seizures, epilepsia, cerebral abscess, encephalopathiesand multiple sclerosis in a sample of an individual wherein the markersare selected from the group consisting of glutathione S transferase Pi(GSTP), fatty-acid-binding protein (FABP), glial fibrillary acidicprotein (GFAP), neuron-specific enolase (NSE), neuromodulin (GAP43),neurofilament protein H (NFH), neurofilament protein M (NFM),neurofilament protein L (NFL), S100B, Tau, spectrin breakdown products,ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1), vascular celladhesion protein 1 (VCAM), serum amyloid A (SAA), Chemokine (C-C motif)ligand 23 (CCL23), peroxiredoxin 1 to 6 and nucleoside diphosphatekinase (NDKA).
 9. Composition comprising or consisting of two or moremarkers wherein the markers are selected from S100B, GSTP, HFABP, VCAM,serum amyloid A (SAA), Chemokine (C-C motif) ligand 23 (CCL23),peroxiredoxin 1 to 6 and NDKA or fragments, variants or mutants thereof.10. Composition according to claim 9 wherein the TBI is mild TBI (mTBI).11. Kit comprising or consisting of two or more markers according toclaim
 1. 12. Assay device comprising or consisting of two or moremarkers according to claim
 1. 13. Assay device according to claim 12which device comprises or consists of a biochip, biomarker panel, acarrier, or a test strip.
 14. Method, according to claim 1 wherein thesensitivity is more than 95%, preferably 100%, and the specificity ismore than 50%, preferably more than 55%, more preferably more than 60%,and more preferably more than 70%.
 15. A method according to claim 1,which makes use of an algorithm.
 16. Composition according to claim 8wherein the sensitivity is more than 95%, preferably 100%, and thespecificity is more than 50%, preferably more than 55%, more preferablymore than 60%, and more preferably more than 70%.
 17. Kit according toclaim 11 wherein the sensitivity is more than 95%, preferably 100%, andthe specificity is more than 50%, preferably more than 55%, morepreferably more than 60%, and more preferably more than 70%.
 18. Assayaccording to claim 12 wherein the sensitivity is more than 95%,preferably 100%, and the specificity is more than 50%, preferably morethan 55%, more preferably more than 60%, and more preferably more than70%.